PROTISTOLOGY European Journal of Protistology 45 (2009) 13–20

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European Journal of PROTISTOLOGY European Journal of Protistology 45 (2009) 13–20 www.elsevier.de/ejop

Pankovaia semitubulata gen. et sp. n. (Microsporidia: Tuzetiidae) from nymphs of mayﬂies Cloeon dipterum L. (Insecta: Ephemeroptera) in Western Siberia Anastasia V. Simakovaa, Yuri S. Tokarevb,Ã, Irma V. Issib aResearch Institute of Biochemistry and Biophysics, Tomsk State University, pr. Lenina 36, Tomsk 634050, Russia bAll-Russian Institute for Plant Protection, Russian Academy of Agricultural Sciences, sh. Podbelskogo 3, St-Petersburg, Pushkin 196608, Russia

Received 16 September 2007; received in revised form 24 April 2008; accepted 30 April 2008

Abstract

The ultrastructure of a new microsporidian, Pankovaia semitubulata gen. et sp. n. (Microsporidia: Tuzetiidae), from the fat body of Cloeon dipterum (L.) (Ephemeroptera: Baetidae) is described. The species is monokaryotic throughout the life cycle, developing in direct contact with the host cell cytoplasm. Sporogonial plasmodium divides into 2–8 sporoblasts. Each sporoblast, then spore, is enclosed in an individual sporophorous vesicle. Fixed and stained spores of the type species P. semitubulata are 3.4 1.9 mm in size. The polaroplast is bipartite (lamellar and vesicular). The polar ﬁlament is isoﬁlar, possessing 6 coils in one row. The following features distinguish the genus Pankovaia from other monokaryotic genera of Tuzetiidae: (a) exospore is composed of multiple irregularly laid tubules with a lengthwise opening, referred to as ‘‘semitubules’’; (b) episporontal space of sporophorous vesicle (SPV) is devoid of secretory formations; (c) SPV envelope is represented by a thin fragile membrane. r 2008 Elsevier GmbH. All rights reserved.

Keywords: Cloeon dipterum; Pankovaia semitubulata gen. et sp. n.; Microsporidia; Ephemeroptera; Ultrastructure

Introduction Plistophora ( ¼ Pleistophora), Gurleya, Stempellia, Tel- omyxa and one undefined species. Thirty seven years Microsporidia of mayflies of Europe and North later, Weiser (1961) extended this list with two species of America have been extensively studied. Ten species of Nosema, two species of Plistophora ( ¼ Pleistophora), these parasites were listed by Kudo (1924) in his three species of Thelohania and a species of a new genus monograph summarizing results of research on micro- Trichoduboscqia. sporidia from all groups of animal hosts. Three of these Based upon ultrastructural features of sporogonial 10 species were attributed to the genus Nosema stages, three Nosema species from mayflies were then according to the taxonomic criteria of that time. The transferred to the new genus Tuzetia (Maurand et al. others were diagnosed as Thelohania (two species), 1971; Maurand 1973). In a review that followed, Larsson (1983) included 6 microsporidian species para- ÃCorresponding author. Tel.: +78 12 377 2923; sitizing Ephemeroptera in the genus Tuzetia. The main fax: +78 12 470 5110. taxonomic features of the Tuzetia genus proposed by the E-mail address: [email protected] (Y.S. Tokarev). author were: (a) monokaryotic nuclear apparatus in all

0932-4739/$ - see front matter r 2008 Elsevier GmbH. All rights reserved. doi:10.1016/j.ejop.2008.04.003 ARTICLE IN PRESS 14 A.V. Simakova et al. / European Journal of Protistology 45 (2009) 13–20 stages of the life cycle; (b) small number of sporoblasts produces 2–4 transitional meront-sporont stages, which (up to 8), resulting from rosette-like sporont fission; (c) give rise to rosette-like sporogonial plasmodia, each individual sporophorous vesicle (SPV) around each dividing into 4–8 sporoblasts, then spores (Figs 3 and 4). spore. Therefore, at the beginning of our studies there Spore development is monomorphic, the stained spores were as many as 20 species of microsporidia in mayflies, being 3.4 mm 1.9 mm (2.6–3.9 1.9–2.2 mm) in size and belonging to 7 genera. However, microsporidia from the oval or oval-cylindrical. On Giemsa-stained slides the mayflies (Insecta: Ephemeroptera) of Russian fauna exospore is visible as a broad blue rim around the were not studied before. spores, suggesting that the uneven surface of the spores The goal of our study is to describe the life cycle and facilitates deposition of the dye. Spores with an extruded morphology of a microsporidian parasite from nymphs polar tube may be observed on the slides (Fig. 4). of Cloeon dipterum in Russia, and to determine its Discharged spores with ejected invasion tubes are taxonomic position using morphological and ultrastruc- observed regularly on fixed smears, suggesting the tural criteria. possibility of parasite’s dissemination due to autoinva- sion within the host organism. Studies of life cycle stages by TEM revealed more Materials and methods details. The meronts are the largest developmental stages (Figs 5–7). Division of merogonial plasmodium produces several, usually 4, spherical meronts, Nymphs of the mayfly Cloeon dipterum were collected 2.6–3.5 mm in size in late stages, with nuclei up to in flood-plain ponds around the town of Tomsk in July 1.8 mm in diameter occupying much of the cell (Fig. 7). 1996. For light microscopy (LM), smears of infected These developmental stages possess nucleoplasm and insect tissues were fixed with methanol for 5 min, stained cytoplasm of low electron density. Nucleoli were not with Giemsa for 24 h (in a modification proposed by seen, but the presence of centriolar plaques indicates Weiser, 1961) and subsequently washed with deionized mitotic activity. water. Digital images were acquired using Carl Zeiss During sporogony each sporont forms a sporogonial Axio 10 Imager M1 with an attached digital camera. plasmodium with 4 to 8 nuclei. Sporont nuclei are Measurements of spores were performed with Carl Zeiss 1.6–1.8 mm in diameter. The sporogonial plasmodium Axiovision software version 4.4.6. divides by rosette-like budding followed by lengthening For transmission electron microscopy (TEM), tissue of each ‘‘rose-petal’’. Electron-dense granular material is pieces were fixed with 2.5% glutaraldehyde solution in accumulated in a spotty pattern on the surface of the 0.1 M cacodylate buffer (pH 7.2) with 4% sucrose for sporogonial plasmodium, making the cell surface to 1–2 h and postfixed with 1% cacodylate-buffered look like a leopard’s skin. These spots are up to 180 nm osmium tetroxide for 1 h. The tissues were dehydrated in height and 100–200 nm in diameter (Fig. 8). in ascending ethanol series and absolute acetone and Division of a sporogonial plasmodium produces embedded in epon-araldite resin. Ultrathin sections were up to 8 sporoblasts (Fig. 9). Later sporoblasts differ stained with 2% uranyl acetate in 50% ethanol and lead from the early ones by higher amounts of electron- citrate for 10–20 min. The material was examined in a dense granules on the surface, denser cytoplasm JEM-100 CX II electron microscope at an accelerating and the presence of nucleoli (Fig. 10). During their voltage of 80 kV. development, sporoblasts change their shape from Repeated screening for microsporidian infection of broad-oval to oval and decrease in size from 4.9 2.6 mayfly populations around Tomsk in 2003–2004, failed to 3.0 1.3 mm. to yield new material, and diagnosis using molecular The next transitional stage from late sporoblast to techniques was not available, restricting our research to young spore is a long-oval cell with smaller size and light and electron microscope analysis. denser cytoplasm as compared to the early sporoblast (Fig. 11). The cytoplasm of the young spore contains primordia of the polar filament and anchoring disc. This Results cell is covered with a thick continuous layer of secretory material, undulating due to its varying thickness, Studies by light microscopy (LM) of the life cycle ranging from 10 to 130 nm (Fig. 11). Cross sections of stages of Pankovaia semitubulata gen. et sp. n. found in fine tubular structures can be seen in the core of the adipose tissue of Cleon dipterum revealed that all stages secretory material. of the life cycle are monokaryotic and develop in direct The mature spores are oval-cylindrical, with a size contact with host cell cytoplasm (Figs 1 and 2). Some of (when fixed and stained) of 2.5–3.2 1.0–1.3 mm(Figs the developmental stages of microsporidia are found 4,5,15). The anchoring disc is a flattened structure under accumulated at the border of the striated muscle cells the polar sac, the latter covering the polaroplast (Figs 15 (Figs 2 and 4). A rosette-like merogonial plasmodium and 16). The polaroplast is bipartite; the anterior ARTICLE IN PRESS A.V. Simakova et al. / European Journal of Protistology 45 (2009) 13–20 15

Figs 1–4. Pankovaia semitubulata sp. n. light microscopy of the Giemsa-stained slides. (1) Adipose tissue cells of the mayﬂy, ﬁlled with meronts with large nuclei. (2) A region of infected tissue at the border of the striated muscle. (3) A binucleate and an octonucleate sporont. (4) Mature spores with intensively stained outer layer of the spore wall. M – meront, Pt – polar tube, S – spore, Sp – sporont, Sm – striated muscle. Scale bars: 10 mm.

horseshoe-shaped (close to circular) region of several They are up to 130 nm high and 60–100 nm wide; the tightly packed lamellae surrounds a region of small length of some of the semitubules reaches 500 nm vesicles filled with inclusions of varied electron density (Fig. 17). Due to the presence of these structures on (Fig. 16). The isofilar polar filament is 150–130 nm in the spore surface, the thickness of the exospore reaches diameter and has 6–6.5 coils in one row (Fig. 15). No 130 nm, similar to the thickness of the endospore. posterior vacuole was found on a series of sections of The SPV envelope in this species is formed only at the mature spores. final phase of sporogony. An additional thin layer of The exospore is a complex structure, composed of moderate electron density appears on small regions of numerous ‘‘gutter-shaped’’ elements. In cross sections, the electron-dense layer surrounding the young spore they possess the shape of a Latin letter ‘‘C’’, while in (Fig. 11). At the phase of spore maturation a single- tangential sections they look like multiple tubules with a layered envelope can be seen tightly bound to the lengthwise opening, irregularly laid on the spore surface, complex protrusions of the exospore (Figs 11,12,15). with their openings outwards (Figs 12–17). Due to these Some regions of this envelope bear tubular structures features, we refer these formations as ‘‘semitubules’’. or form long protrusions into the host cell cytoplasm ARTICLE IN PRESS 16 A.V. Simakova et al. / European Journal of Protistology 45 (2009) 13–20

Figs 5–7. Merogonial stages of Pankovaia semitubulata sp. n. (TEM) (5) Section of adipose tissue of mayﬂies with different developmental stages of microsporidia. (6) Late meronts with large nuclei. (7) Rosette-like merogonial plasmodium with dividing nuclei. Cp – centriolar plaque, Cu – cuticle; Hy – hypoderm, M – meront, Mpl – merogonial plasmodium, N – nucleus, S – spore, Sb – sporoblast, Sp – sporont. Scale bars: Fig. 5–5 mm; Figs. 6, 7–1 mm.

(Fig. 12). The envelope is gradually detached from the and hardly visible around the mature spore. Tubular spore, forming an electron-translucent SPV cavity structures or ﬁlaments connecting the exospore with the (Figs 13 and 14). The vesicle envelope is ephemeral SPV envelope, as well as the reticulate structure of the ARTICLE IN PRESS A.V. Simakova et al. / European Journal of Protistology 45 (2009) 13–20 17

Figs 8–11. Sporogonial stages of Pankovaia semitubulata sp. n. (TEM) (8) Rosette-like sporogonial plasmodium with abundant electron-dense granules on the surface. (9–10) Sporoblasts with secretion material accumulated in a spotty pattern. (11) Transitional stage late sporoblast to young spore. Adp – primordium of anchoring disc. Dc – dense surface coat. M – meront. N – nucleus. Nu – nucleolus. Sg – secretion granules. Spl – sporogonial plasmodium. Spv – sporophorous vesicle. Sy – young spore. Scale bar: Figs. 8–11–1 mm.

SPV wall itself, typical of Tuzetia species, are absent genera, namely Tuzetia, Alfvenia, Janacekia, Nelliemelba, from this microsporidian. Lanatospora, Trichotuzetia and Paratuzetia (Larsson 1983; Poddubnaya et al. 2006; Vavra et al. 1997; Voronin 1989, 2001). Only three of these genera are monokaryotic during late merogony and sporogony, as Discussion well as the newly established genus Pankovaia. There- fore these genera, Tuzetia, Lanatospora and Trichotuze- This is the ﬁrst description of a microsporidian tia, need to be compared with the type species of the parasite infecting a mayﬂy from Russia. This micro- genus described in the present work. sporidium belongs to a new genus and species, which we These three genera share following similar features in have named Pankovaia semitubulata. cell morphology of the sporogonial stage: (a) rosette-like Pankovaia semitubulata has an individual SPV sheath division of sporonts with simultaneous production of around each spore and therefore should be attributed to sporoblasts enclosed within an individual SPV sheath; the family Tuzetiidae (see Larsson 1983 for the family (b) accumulation of discrete electron-dense material on diagnosis). This family at present incorporates seven the sporoblast surface; (c) epispore structures (tubules, ARTICLE IN PRESS 18 A.V. Simakova et al. / European Journal of Protistology 45 (2009) 13–20

Figs 12–17. Sporophorous vesicles and spores of Pankovaia semitubulata sp. n. (TEM) (12–14) Formation of episporontal space of SPV. (15) Mature spore with cross-section of ‘‘semitubular’’ structures of exospore. (16) Anterior part of the spore with ﬂattened anchoring disc and bipartite polaroplast, in which the anterior horseshoe-like lamellar part surrounds the posterior vesicular one. (17) Longitudinal section of spore wall showing semitubular structure of the exospore. Ad – anchoring disc. En – endospore. Ex – exospore. Ht – half-tube sectioned longitudinally. N – nucleus. Pp1, Pp2 – anterior and posterior parts of polaroplast. Pt – polar ﬁlament. Ss – semitubular structures. Sve – membrane of SPV. Scale bar: Figs. 12–17–0.5 mm.

fibrils or fleecy layer) filling the episporontal space of the exospore is thin, the SPV cavity is partially filled with SPV. secretory tubules, the polar filament is isofilar and the Distinctions between these genera are as follows: in SPV sheath is reticular; in Trichotuzetia, the sporont Tuzetia, the sporont gives rise to 2–8 sporoblasts, the gives rise to 20–30 sporoblasts, the exospore is 4-layered, ARTICLE IN PRESS A.V. Simakova et al. / European Journal of Protistology 45 (2009) 13–20 19 the SPV cavity is completely filled with fibrils, and the A few words about the composition of the Tuzetiidae polar filament is isofilar; in Lanatospora, 6–16 sporoblasts are necessary. In our opinion, there are currently four are formed, the exospore is 2-layered, covered by blister- genera that undoubtedly belong to this family: Tuzetia like structures filled with a fleecy material, a typical SPV is (Larsson 1983), Lanatospora (Voronin 1989), Trichotu- absent, and the polar filament is heterofilar. zetia (Vavra et al. 1997) and the newly established genus Thetypespeciesofthemicrosporidian described in the Pankovaia. They are characterized by development in present work is distinguished from these genera by the direct contact with the host cell cytoplasm, monokar- following features: (a) a continuous layer of amorphous yotic developmental stages, rosette-like fission of mer- secreted material on the surface of late sporoblasts and onts and sporonts (as a rule; less frequently, ribbon-like young spores; (b) the texture of the exospore, consisting of or chain fission is observed), spores of the same type, semitubular structures (see above); (c) the absence of formed within individual SPV, and isofilar polar secretory formations in the episporontal space of the SPV. filaments. The position of the other four genera An exospore with such a complicated structure is not (Janacekia, Alfvenia, Nelliemelba and Paratuzetia) form- known for other genera of the family Tuzetiidae, or for ing diplokaryotic developmental stages and attributed other microsporidia. For these reasons, we propose to by Larsson (1983), Poddubnaya et al. (2006) to the establish the new genus Pankovaia with the type species of Tuzetiidae family, is uncertain. The former author Pankovaia semitubulata, described in the present work. himself regarded their position as temporary: ‘‘In the The finding of a new microsporidian species para- future it will probably be necessary to split the genera of sitizing Cloeon dipterum L. in Western Siberia is the Tuzetiidae into more than one family’’ (Larsson interesting for two reasons. First, this is the first record 1983, p. 354). The same should be applied to the genus of microsporidia from mayflies in Russia. Second, it is Paratuzetia as well. an addition to the list of parasites found in this widely distributed palearctic insect. Two microsporidia have Diagnosis of Pankovaia gen. n. been described from Cloeon dipterum in Europe. A discovery of new microsporidian parasitizing the same Monomorphic and monokaryotic throughout the life mayfly is an indication of the originality of parasitic cycle. All stages develop in direct contact with host-cell fauna in the Asian part of Russia. cytoplasm. Merogonial and sporogonial plasmodia fis- It is noteworthy that the presence of more than one sion is performed by a rosette-like budding with microsporidian species is often revealed in intensively production of 2–4 late meronts and 4–8 sporoblasts, studied insect and crustacean hosts. For example, there respectively. During sporogony, amorphous electron- are ten species of microsporidia in Chironomus plumosus dense material is arranged in a spotty pattern on the (Chironomidae), eight and six species in the cladoceran surface of rosette-like sporonts, transforming into a crustaceans Daphnia pulex and D. magna, respectively. continuous layer on the surface of sporoblasts and young It is probable that the mayfly Cloeon dipterum possesses spores. Spores possess a bipartite polaroplast and isofilar various sets of parasites in different parts of its vast polar filament. The exospore is single-layered, with a distribution area. complicated texture. The SPV envelope is represented by A certain level of similarity is found between a thin fragile membrane. The SPV episporontal space is Pankovaia semitubulata and Tuzetia weidneri (Canning devoid of secretory inclusions. Parasites of Ephemer- et al. 2002). The spore wall of the latter is decorated with optera. Type species: Pankovaia semitubulata. ridges and its attribution to the genus Tuzetia is supplied Etymology. Named in honour of Tamara F. Pankova, with the following stipulation: ‘‘We have tentatively an associate professor of Tomsk State University, who assigned the species to the genus Tuzetia in spite of the initiated research of insect microsporidia in Western difference which exists between the episporontal secre- Siberia. tions and although the prominent system of ridges on the spore wall is a new feature for the genus’’ (Canning et al. 2002, p. 73). Diagnosis of Pankovaia semitubulata sp. n. T. weidneri and P. semitubulata possess the following Type host: mayfly Cloeon dipterum (L.) (Ephemer- similar features: (a) complicated composition of the optera, Baetidae). spore wall; (b) absence of any secretory material in the Localization: adipose tissue. episporontal space of the SPV; (c) typical membranous Type locality: vicinity of Tomsk, Western Siberia. structure of the SPV sheath. However, meronts in T. weidneri are ribbon-like in contrast to those of P. semitubulata. Confirmation of a close relationship Developmental stages: Merogony and sporogony with between these two species of microsporidia will be characteristics of the genus. Spores are oval-cylindrical, possible only after comparison of their respective rDNA 3.4 1.9 mm in size after fixation. The exospore is nucleotide sequences. composed of multiple irregularly laid tubules with a ARTICLE IN PRESS 20 A.V. Simakova et al. / European Journal of Protistology 45 (2009) 13–20 lengthwise opening, referred to as ‘‘semitubules’’. The Larsson, R., 1983. A revisionary study of the taxon Tuzetia polaroplast is bipartite (lamellar and vesicular). The Maurand, Fize, Fenwick and Michel, 1971, and related isofilar filament possesses 6–6.5 coils. The SPV envelope forms (Microspora, Tuzetiidae). Protistologica 19, is ephemeral, virtually disappearing in mature spores. 323–355. Maurand, J., 1973. Recherches biologiques sur les Micro- sporidies des larves des Simulies. These. Universite des Etymology Sciences et Techniques du Languedoc, Academie de The species name reflects the shape of complicated Montpellier, France, pp. 1–199. structures (semitubules), composing the exospore in Maurand, J., Fize, A., Fenwick, B., Michel, R., 1971. Etude au mature spores. microscope electronique de Nosema infirmum Kudo 1921. Microsporidie parasite d’un Copepode Cyclopoide, crea- tion du genre nouveau Tuzetia a propos de cette espece. Acknowledgements Protistologica 7, 221–225. Poddubnaya, L.G., Tokarev, Y.S., Issi, I.V., 2006. A new microsporidium Paratuzetia kupermani gen et sp. n. The research is supported by the Russian Foundation (Microsporidia), a hyperparasite of the procercoid of the for Basic Research, Grant nos. 07-04-00269 and 07-04- cestode Khawia armeniaca Chol. 1915 (Cestoda, Caryo- 00468, and a grant from the President of Russian phyllidea). Protistology 4, 269–277. Federation no. MK-653.2007.4. Vavra, J., Larsson, R., Baker, M., 1997. Light and electron microscopic cytology of Trichotuzetia guttata gen et sp. n. (Microspora, Tuzetiidae), a microsporidian parasite of References Cyclops vicinus Uljanin, 1875 (Crustacea, Decapoda). Arch. Protistenkd. 147, 293–306. Canning, E.U., Curry, A., Overstreet, R.M., 2002. Ultra- Voronin, V.N., 1989. The ultrastructure of Lanatospora structure of Tuzetia weidneri sp n. (Microsporidia: Tuzetii- macrocyclopis (Protozoa: Microsporidia) from the cyclope dae) in skeletal muscle of Litopenaeus setiferus and Macrocyclops albidus (Jur) (Crustacea, Copepoda). Arch. Farfantepenaeus aztecus (Crustacea: Decapoda) and new Protistenkd. 137, 356–357. data on Perezia nelsoni (Microsporidia: Pereziidae) in L. Voronin, V.N., 2001. Macrotaxonomy of the phylum Micro- setiferus. Acta Protozool. 41, 63–77. sporidia. Parazitologya 35, 35–44. Kudo, R., 1924. A biologic and taxonomic study of the Weiser, J., 1961. Die mikrosporidien als parasiten der insekten. Microsporidia. Illin. Biol. Monogr. 9, 1–268. Monogr. Angew. Entomol. 17, 1–149.